The present invention relates, in general, to recombinant viable chimeric flaviviruses, and to vaccines for dengue virus and other flaviviruses, including tick-borne encephalitis virus (TBEV). The invention further relates to cDNA sequences encoding the RNA transcripts to direct the production of recombinant dengue type 4 virus, recombinant mutant dengue type 4 virus chimeric dengue viruses, chimeric dengue viruses incorporating mutations to recombinant DNA fragments generated therefrom, and the cells transformed therewith.
This invention further relates to vaccines produced from recombinant DNA. Specifically, this invention relates to vaccines for dengue virus encephalitis from recombinant DNA. The strategy used in this invention is also applicable to the development of subunit vaccines against other important flaviviruses such as Japanese B encephalitis virus, and the tick-borne encephalitis viruses for which routine vaccines are generally not available.
The family Flaviviridae includes approximately 60 enveloped, positive strand RNA viruses, most of which are transmitted by an insect vector. Many members of this family cause significant public health problems in different regions of the world (Monath, T. P. (1986) In: The Togaviridae and Flaviviridae. S. Schlesinger et al., eds. pp. 375-440. Plenum Press, New York). The genome of all flaviviruses sequenced thus far has the same gene order: 5xe2x80x2-C-preM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3xe2x80x2 in which the first three genes code for the structural proteins the capsid (C), the premembrane protein (pre M) and the envelope protein (E).
Dengue is a mosquito-borne viral disease which occurs in tropical and sub-tropical regions throughout the world. The dengue virus subgroup causes more human disease than any other member of the flavivirus family. Dengue is characterized by fever, rash, severe headache and joint pain. Its mortality rate is low. However, over the past few decades, a more severe form of dengue, characterized by hemorrhage and shock (dengue hemorrhagic fever/dengue shock syndrome; DHF/DSS) has been observed with increasing frequency in children and young adults. DHF/DSS occurs most often during dengue virus infection in individuals previously infected with another dengue virus serotype. This has led to the suggestion that immune enhancement of viral replication plays a role in the pathogenesis of the more severe form of disease (Halstead, S. B. (1988) Science 239, 476-481).
Dengue epidemics are a major public health problem in many tropical and subtropical areas where the vector mosquito species are abundant. Despite 40 years of intensive research, safe and effective vaccines for dengue virus disease are not available. The WHO has assigned dengue virus as a high priority target for accelerated research and vaccine development.
Soon after their isolation in 1944, dengue viruses were passaged repeatedly in mouse brain, resulting in the selection of mouse neurovirulent mutants (Sabin, A. B. (1952) Amer. J. Trop. Med. Hyg. 1:30-50). Interestingly, studies performed in volunteers showed that mouse brain-adapted neurovirulent mutants of three strains of type 1 or type 2 dengue virus were attenuated, but still immunogenic for humans (Sabin, A. B. (1952) Amer. J. Trop. Med. Hyg. 1:30-50; Sabin, A. B. (1955) Amer. J. Trop. Med. Hyg. 4:198-207; Sabin, A. B. (1955) Amer. J. Trop. Med. Hyg. 4:198-207; Schlesinger, R. W. et al. (1956) J. Immunol. 77:352-364; Wisseman, C. L. et al. (1963) Amer. J. Trop. Med. 12:620-623). However, the mutants were not developed further as candidate vaccine strains because of concern for mouse brain antigens in the vaccine preparations. Since that time, virus mutants that: (i) exhibited the small plaque size phenotype, and/or (ii) were temperature sensitive, and/or (iii) were adapted to cell cultures derived from an unnatural host (i.e., host range mutants), have been selected and evaluated as candidates for inclusion in a live attenuated virus vaccine (Harrison, V. R. et al. (1977) Infec. Immun. 18:151-156; Hoke, C. H. et al. (1990) Am. J. Trop. Med. Hyg. 43:219-226; Bhamarapravati, N. et al. (1987) Bull. WHO. 65:189-195). However, despite 25 years of such efforts, safe, effective dengue vaccines are still not available for general use. Inactivated whole dengue virus vaccines have been shown to be insufficiently immunogenic. Live virus vaccines attenuated by serial passage in cell culture have suffered from genetic instability under attenuation or poor immunogenicity. The present invention represents a technical breakthrough by providing chimeric dengue and flavivirus vaccines.
These four serotypes of dengue viruses (type 1 to type 4) are distinguishable by plaque reduction neutralization using serotype-specific monoclonal antibodies and by less specific tests using polyclonal sera (Bankcroft, W. M. et al. (1979) Pan Am. Hlth. Org. Sci. Publ. 375:175-178; Henchal, E. A. et al. (1982) Am. J. Trop. Med. Hyg. 31:548-555). The existence of serotypes was first discovered during early studies in human volunteers, which showed that infection with one dengue serotype induced durable homotypic immunity, whereas heterotypic immunity lasted only 3 to 5 months (Sabin, A. B. (1 952) Amer. J. Trop. Med. Hyg. 1:30-50). An effective dengue vaccine that contains all four serotypes in order to induce broad immunity to dengue viruses in general would help to preclude the occurrence of DHF/DSS.
The complete nucleotide sequences have been determined for dengue virus types 3 and 4 and several strains of type 2 virus including the mouse-neurovirulent New Guinea C, however, only the 5xe2x80x2 portion of the type 1 virus genome has been sequenced (Mackow, E. et al. (1987) Virology 159:217-228; Zhao, B. et al. (1986) Virology 155:77-88; Osatomi, K. and Sumiyoshi, H. (1990) Virology 176:643-647; Irie, A. et al. (1989) Gene 75:197-211; Mason, P. W. et al. (1987) Virology 161:262-267; Hahn, Y. S. et al. (1988) Virology 162:167-180). The results of these studies indicate that the four dengue virus serotypes share a common genome organization. The genome of the dengue type 4 Caribbean strain 814669 was found to contain 10646 nucleotides (Mackow, E. et al. (1987) Virology 159:217-228; Zhao, B. et al. (1986) Virology 155:77-88). The first 101 nucleotides at the 5xe2x80x2 end and the last 384 at the 3xe2x80x2 end are non-coding. The remaining sequence codes for a 3386 amino-acid polyprotein which includes the three structural proteins, namely, capsid (C), premembrane (pre-M), and envelope (E), at its N-terminus, followed by seven non-structural proteins in the order, provided above, that is consistent with all Flavivirus genomes identified thus far. The polyprotein is processed to generate 11 or more viral proteins by cell signal peptidase(s) and by viral proteases (Markoff, L. (1989) J. Virol, 63:3345-3352; Falgout, B. et al. (1989) J. Virol, 63:1852-1860; Falgout, B. et al. (1991) J. Virol. 65:2467-2476; Hori, H. and Lai, C. J. (1990) J. Virol. 64:4573-4577).
Previously we constructed a full-length dengue virus cDNA that could serve as the template for transcription of infectious RNA. We have obtained stably cloned full-length dengue virus cDNA and in vitro RNA transcripts derived from the DNA template were shown to be infectious for cells in culture. However, this infectious construct and infectious RNA transcripts generated therefrom are pathogenic. Moreover, the attenuated dengue viruses generated thus far are genetically unstable and have the potential to revert back to a pathogenic form over time. Yet, attenuated viruses are desirable since they are generally known to provided long-lasting immunity. Therefore, modifications to this construct or to chimeric constructs that then direct the production of a less pathogenic virus would be a considerable advance to attenuated flavivirus vaccine technology. Accordingly, we have constructed a series of deletions in the 3xe2x80x2 non-coding region of cDNA, as disclosed herein, and have recovered viable dengue virus mutants for analysis of growth characteristics.
Other members of the Flavivirus family are also pathogenic. Examples include tick-borne encephalitis virus and Japanese Encephalitis Virus. Like attenuated dengue virus vaccines, attenuated tick-borne encephalitis virus (TBEV) virus has tended to be genetically unstable and poorly immunogenic. Therefore, other attenuated flavivirus vaccines would also be a considerable advance in the art. Thus, this invention additionally employs modified full-length recombinant cDNA constructs of dengue virus or another flavivirus as a framework for gene manipulation and chimeric virus development for the production of vaccines to other Flaviviruses.
Tick-borne encephalitis virus (TBEV) is transmitted exclusively by ticks and can be divided into two serologically distinguishable subtypes: the Eastern subtype (prototype strain Sofjin), prevalent in Siberian and Far Eastern regions of Russia, and the Western subtype (prototype strain Neudorfl), common in eastern and central Europe. TBEV causes a serious encephalitic illness with a mortality rate ranging from 1 to 30%. For a review of TBEV see Calisher, et al. (J. Gen. Virol 70: 37-43). Currently, an experimental TBE vaccine produced by formalin inactivation of TBEV is available, but this vaccine has several limitations. For example, the vaccine is not sufficiently immunogenic, therefore repeated vaccinations are required to generate a protective immune response. Even when antibody responses to the vaccine are present, the vaccine fails to provide protective responses to the virus in 20% of the population. Therefore, there remains a need for an improved TBEV vaccine.
Dengue viruses continue to cause major epidemics throughout the tropical and subtropical regions of the world. Despite many years of research effort, an effective vaccine is not available. The predominant disease associated with dengue viral infection is a debilitating illness known as dengue fever. Less frequently, dengue virus causes a hemorrhagic shock syndrome in young children, which has a very high mortality rate. Thus, control of dengue fever and dengue hemorrhagic shock is a major global concern. Consequently, the WHO has designated the dengue viruses as one of five high priority targets for accelerated vaccine development. The industry is lacking a vaccine formed from a genetically engineered dengue protein.
In one aspect, the present invention provides a recombinant DNA construct containing nucleic acid derived from at least two flaviviruses. The construct includes a region of nucleic acid operably encoding no more than two structural proteins from tick-borne encephalitis virus (TBEV). This region is operably linked to a region of nucleic acid encoding structural proteins from a flavivirus other than TBEV. Preferably, the construct includes a region of nucleic acid operably encoding flavivirus capsid protein operably linked to the region of nucleic acid operably encoding no more than two structural proteins from tick-borne encephalitis virus. The regions of nucleic acid encoding capsid protein and non-structural proteins are preferably from dengue virus, such as dengue type 4 virus. In a preferred form, the construct contains at least one mutation in the nucleic acid derived from at least two flaviviruses. In one embodiment of this preferred form, the mutation ablates NS1(1) protein glycosylation. In another embodiment of this form, the mutation prevents production of the mature flavivirus membrane protein. The mutation preferably affects viral growth rate. The invention also includes RNA transcripts corresponding to these recombinant DNA constructs within the present invention.
In another aspect of the present invention, there is provided a chimeric virus having a genome derived from a flavivirus. This chimeric virus include a region of nucleic acid encoding no more than two structural proteins from tick-borne encephalitis virus, and a region of nucleic acid encoding nonstructural proteins from another flavivirus. Preferably, the virus includes a region of nucleic acid encoding capsid protein from the other members of the flavivirus family. The nucleic acid from tick-borne encephalitis virus can encode any of a number of proteins, such as pre-membrane protein and/or the envelope protein. The nucleic acid encoding nonstructural proteins from another flavivirus is preferably from dengue virus, such as type 4 dengue virus. In one embodiment, the chimeric virus of this aspect of the invention contains at least one mutation in the nucleic acid. This mutation can take any of a number of forms, such as a mutation that ablates NS1(1) protein glycosylation, or one that prevents production of the mature flavivirus membrane protein. The mutation preferably affects viral growth rate.
Another aspect of the present invention provides a chimeric virus that includes a region of nucleic acid encoding the premembrane and envelope proteins of tick-borne encephalitis virus, and a region of nucleic acid encoding the capsid protein from another flavivirus, and a region of nucleic acid encoding non-structural proteins from the same flavivirus. The other flavivirus can be, for example, a dengue virus, such as dengue virus type 4. In this aspect of the invention, a preferred form of the chimeric virus includes at least one mutation in the region of nucleic acid encoding premembrane and envelope protein of tick-borne encephalitis virus. The mutation can be one that prevents production of the mature flavivirus membrane protein. In another preferred form, the chimeric virus includes at least one mutation in the regions of nucleic acid from the other flavivirus. This mutation can be one that ablates NS1(1) protein glycosylation.
In another aspect, the invention provides a vaccine for humans against tick-borne encephalitis comprising a chimeric virus of the present invention that is capable of generating a protective immune response in a vertebrate. Thus, the invention also includes a method for preparing a vaccine for humans against a flavivirus comprising preparing a DNA construct operably encoding the premembrane and envelope protein from the flavivirus and the capsid and nonstructural protein from dengue virus, generating infectious RNA transcripts from the DNA construct, introducing the RNA transcripts into a cell, expressing the RNA transcripts in the cell, harvesting the virus from the cells, testing the virus in a vertebrate, and inoculating the humans with the virus. The preparing step can include introducing mutations into the DNA construct.
A preferred aspect of the present invention provides a chimeric virus that includes region of nucleic acid encoding the premembrane and envelope proteins of Japanese encephalitis virus, and a region of nucleic acid encoding the capsid protein from another flavivirus, and a region of nucleic acid encoding non-structural proteins from the same flavivirus, such as dengue virus.
Another aspect of the present invention provides a chimeric virus having an RNA genome. This genome includes a region of nucleic acid operatively encoding non-structural protein of type 4 dengue virus and a region of nucleic acid operatively encoding structural protein of a type 1 dengue virus, type 2 dengue virus, type 3 dengue virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, or another flavivirus. In one embodiment of the invention, the genome of the chimeric virus is substantially free from nucleic acid operatively encoding type 4 dengue virus structural protein. Preferred embodiments of the chimeric virus can include p2A(D1 WP) or p2A(D2 NGC) RNA. Preferably, the chimeric virus includes a region of nucleic acid operatively encoding non-structural protein of type 1 dengue virus, type 2 dengue virus, or type 3 dengue virus, and also includes a region of nucleic acid operatively encoding structural protein of type 1 dengue virus, type 2 dengue virus, type 3 dengue virus, type 4 dengue virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, or another flavivirus. The nucleic acid operatively encoding non-structural protein is preferably from a different virus than the region of nucleic acid operatively encoding non-structural protein.
Another aspect of the invention provides a chimeric virus having an RNA genome. This genome includes a region of nucleic acid operatively encoding non-structural protein of yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus or another flavivirus, and a region of nucleic acid operatively encoding structural protein of type 1 dengue virus, type 2 dengue virus, type 3 dengue virus, type 4 dengue virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, or another flavivirus. The region of nucleic acid operatively encoding structural protein is preferably from a different virus than the region of nucleic acid operatively encoding non-structural protein.
Yet another aspect of the invention provides a vaccine, capable of generating a protective immune response to a virus in a vertebrate. This vaccine includes a safe and immunologically effective amount of any of the viruses of the present invention and a pharmaceutically and immunologically acceptable carrier.
Still another aspect of the invention provides another vaccine that is capable of generating a protective immune response to a virus in a vertebrate. In this aspect of the invention, the vaccine includes a safe and immunologically effective amount of the following viruses: a) a chimeric virus wherein the genome of the virus includes a region of nucleic acid encoding non-structural protein of type 4 dengue virus and a region of nucleic acid encoding structural protein of type 1 dengue virus, b) a chimeric virus, wherein the genome of the virus includes a region of nucleic acid encoding non-structural protein of type 4 dengue virus and a region of nucleic acid encoding structural protein of type 2 dengue virus, c) a chimeric virus, wherein the genome of the virus includes a region of nucleic acid encoding non-structural protein of type 4 dengue virus and a region of nucleic acid encoding structural protein of type 3 dengue virus, and d) an attenuated type 4 dengue virus.
A further aspect of the present invention provides a DNA segment that includes a non-structural region of type 4 dengue virus, and a structural region from one of the following viruses: type 1 dengue virus, type 2 dengue virus, type 3 dengue virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and another flavivirus. In a preferred embodiment of the invention, the DNA segment contains a promoter operably linked to the structural and nonstructural regions. In another preferred embodiment, the DNA segment contains a promoter which is a SP6 or T7 promoter. In yet another preferred embodiment, the DNA segment is p2A(D1 WP) or p2A(D2 NGC).
Other aspects of the invention provide other chimeric DNA segments. The DNA segments of these aspects of the invention include a non-structural region of type 1 dengue virus, type 2 dengue virus or type 3 dengue virus. These DNA segments further include a structural region from one of the following viruses: type 1 dengue virus, type 2 dengue virus, type 3 dengue virus type, 4 dengue virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and a flavivirus, with the proviso that the structural region and the non-structural region are not from the same virus.
A further aspect of the invention provides a DNA segment that includes a non-structural region from one of the following viruses: yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and another flavivirus, and a structural region from one of the following viruses: type 1 dengue virus, type 2 dengue virus, type 4 dengue virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and another flavivirus wherein the structural region is from a different virus than the non-structural region.
Another aspect of the invention provides a DNA segment comprising a non-structural region or a portion thereof of a flavivirus, and a structural region or a portion thereof from a different flavivirus.
Still another aspect of the invention provides a vaccine, capable of generating a protective immune response to a virus in a vertebrate. This vaccine includes a safe and immunologically effective amount of a chimeric virus. The genome of the virus includes nucleic acid operably encoding the structural proteins from a tick-borne encephalitis virus and nucleic acid operably encoding the non-structural protein from a dengue virus.
Yet another aspect of the invention provides another chimeric virus. In this aspect, the chimeric virus has a genome of the virus operably encoding tick-borne encephalitis structural protein and dengue type 4 virus non-structural protein. In a preferred embodiment, the structural proteins of the virus are derived from tick-borne encephalitis virus.
Still another aspect of the invention provides a segment of DNA operably encoding tick-borne encephalitis structural protein and dengue type 4 virus non-structural protein.
A further aspect of the invention provides an isolated DNA fragment that encodes infectious dengue type 4 viral RNA. Still another aspect of the invention provides a recombinant DNA construct comprising the DNA fragment of the present invention, and a vector. In a preferred embodiment, the vector is a plasmid. A further aspect of the invention includes a host cell stably transformed with a recombinant DNA construct according to the present invention in a manner allowing expression of the DNA fragment. In a preferred embodiment, the host cell is a prokaryotic cell.
Another aspect of the invention provides a method for producing mutants of dengue type 4 virus. This method includes the steps of (i) introducing mutations into the genome of dengue type 4 virus by site-directed mutagenesis,(ii) recovering infectious dengue type 4 viruses harboring the mutations, and (iii) evaluating recovered viruses. In one embodiment of this method, the recovered viruses are evaluated for attenuated or a virulent phenotype.
Still another aspect of the invention provides a vaccine for humans against dengue type 4 virus. This vaccine includes an a virulent dengue type 4 virus, in an amount sufficient to induce immunization against the disease, and a pharmaceutically acceptable carrier. In a preferred embodiment, the a virulent dengue type 4 virus is obtained by engineering mutations at strategic regions in the viral genome.
Yet another aspect of the present invention provides a DNA fragment that encodes chimeric flaviviral RNA. The chimeric RNA includes, for example, a member selected from the group consisting of dengue type 1, dengue type 2 and dengue type 3.
Still another aspect of the invention provides a method for construction of chimeric dengue viruses. This method includes replacing DNA fragments of the dengue type 4 virus DNA according to the method of the present invention with corresponding genes en bloc, or a fraction thereof, from a different flavivirus. In a preferred embodiment, the different flavivirus is one of the following viruses: dengue type 1, dengue type 2 and dengue type 3. In another preferred embodiment, the dengue type 4 virus contains at least one mutation.
The present invention includes another aspect which provides a vaccine for humans against dengue virus. In this aspect, the invention includes an infectious chimeric virus, in an amount sufficient to induce immunization against the disease, and a pharmaceutically acceptable carrier. The chimeric virus is derived from, for example, dengue type 1, dengue type 2, dengue type 3 or dengue type 4 virus.
Yet another aspect of the invention provides a recombinant DNA construct comprising the DNA fragment of the present invention, and a vector. In one preferred embodiment, the vector is a plasmid. Still another aspect of the present invention provides a host cell stably transformed with the recombinant DNA construct according to the method of the present invention, in a manner allowing expression of the DNA. In a preferred embodiment, the host cell is a prokaryotic cell.
Still another aspect of the invention provides a DNA fragment that encodes a dengue type 4 viral RNA, wherein the DNA fragment contains a substitution mutation in the sequence encoding one or more of eight amino acids at the C terminus of NS1 of the cleavage site of the non-structural protein NS1-NS2A.
A further aspect of the invention provides a DNA fragment that encodes a dengue type 4 viral RNA, wherein the DNA fragment contains a substitution at the site encoding glycine, which site is at position +1 following the cleavage site of the non-structural protein NS1-NS2A.
A still further aspect of the invention provides a DNA fragment that encodes a dengue type 4 viral RNA, wherein the DNA fragment contains a deletion in the 3xe2x80x2-noncoding region. In a preferred embodiment, the deletion is between 30 and 202 nucleotides in length.
Another aspect of the invention provides a recombinant DNA construct including any of the DNA fragments of the present invention and a vector. Still another aspect of the invention provides an infectious RNA transcript of a DNA fragment of the present invention. A further aspect of the invention provides a host cell transfected with the DNA constructs of the present invention in a manner allowing expression of the DNA fragment. Such a host cell can be a mammalian or an insect cell.
Still another aspect of the invention provides a vaccine for humans against dengue type 4 virus. In this aspect of the invention, the vaccine includes a mutant dengue type 4 virus exhibiting reduced virulence, in an amount sufficient to induce immunization against the disease. In a preferred embodiment, the mutant dengue type 4 virus is obtained by engineering a deletion mutation in the 3xe2x80x2-noncoding region in the viral genome. In another preferred embodiment, the mutant dengue type 4 virus is obtained by engineering a substitution mutation in the viral DNA sequence encoding one or more of eight amino acids at the C terminus of NS1 of the cleavage site of the non-structural protein NS1-NS2A. In yet another preferred embodiment, the mutant dengue type 4 virus is obtained by engineering a substitution at the viral DNA site encoding glycine, which site is at position +1 following the cleavage site of the non-structural protein NS1-NS2A.
Still another aspect of the invention provides a method for construction of chimeric dengue viruses comprising replacing DNA fragments of the dengue type 4 virus DNA with corresponding genes en bloc, or a fraction thereof, from a different flavivirus. In a preferred embodiment, the different flavivirus is one of: dengue type 1, dengue type 2, dengue type 3, Japanese encephalitis and tick-borne encephalitis virus. In another preferred embodiment, the method for construction of chimeric dengue viruses includes replacing DNA fragments of the dengue type 4 virus DNA according to the present invention, with corresponding genes en bloc, or a fraction thereof, from a different flavivirus. Preferably, the flavivirus is selected from the group consisting of dengue type 1, dengue type 2, dengue type 3 and Japanese encephalitis. Also preferably, the method for construction of chimeric dengue viruses includes replacing DNA fragments of the dengue type 4 virus DNA according to the present invention, with corresponding genes en bloc, or a fraction thereof, from a different flavivirus. In the preferred method, the flavivirus is selected from the group consisting of dengue type 1, dengue type 2, dengue type 3, Japanese encephalitis and tick-borne encephalitis virus.
One additional aspect of the invention provides a vaccine against dengue virus that is administered in an amount sufficient to induce immunization against the disease. The vaccine includes a chimeric virus exhibiting reduced virulence, wherein the chimeric virus contains a DNA fragment that encodes a dengue type 4 viral RNA containing a deletion in the 3xe2x80x2-non-encoding region. In a preferred embodiment, the chimeric virus is selected from the group consisting of dengue type 2, dengue type 3 and dengue type 4 virus.
A further aspect of the invention is a baculovirus having a 4.0 kilo-base recombinant sequence dengue cDNA sequence. The sequence encodes dengue virus capsid protein, pre-matrix protein, envelope glycoprotein, and NS1 and NS2a nonstructural proteins. The invention includes a vaccine and a method to produce that vaccine.
Further aspects of the present invention will become apparent to those of ordinary skill in the art upon reference to the ensuing description of the invention.